![]() ![]() ![]() ![]() Background levels of staining were delineated using gates positioned to include 98% of the control cells. Positive and negative populations of cells were determined using unreactive isotype-matched mAbs (BD Pharmingen) as controls for background staining. Cells with the forward and side light scatter properties of lymphocytes were analyzed with fluorescence intensity shown on a four-decade log scale. Low angle and orthogonal light scatters were used to exclude dead cells and debris. A minimum of 10,000 events were collected per sample, and data were analyzed with CellQuest (version 3.1) (BD Biosciences). After washing and fixation in PBS/1% formaldehyde, the cells were analyzed using a single laser FACScan flow cytometer (BD Biosciences). V H3 + cells were identified using biotin-conjugated SpA and FITC-labeled anti-CD45R/B220 Ab. Anti-human CD19-FITC mAbs (J14.119) was provided by Immunotech and anti-mouse CD19-PE mAbs (6D5) by Clinisciences. All those mAbs and their corresponding isotype controls were purchased from BD Pharmingen. Cell phenotype was determined using the following Abs: anti-mouse CD5-PE (53-7.3), anti-mouse CD45R/B220-FITC or anti-mouse CD45R/B220-C圜hrome (RA3-6B2), anti-mouse CD11b-PE (M1/70), anti-mouse CD4-PE (H129.19), anti-mouse CD8-FITC (53-6.7), anti-mouse CD43-FITC (S7), anti-mouse CD21/CD35-FITC (7G6), anti-mouse CD23-PE (B3B4), anti-human IgM-FITC or anti-human IgM-PE (G20-127), and anti-human IgD-FITC (IA6-2). Following washes, when necessary, biotin-labeled Abs were revealed by streptavidin-C圜hrome (BD Pharmingen). Leukocytes (0.5 × 10 6) were stained at 4☌ using predetermined optimal concentrations of different combinations of fluorochrome-labeled Abs for 30 min. Immunofluorescence analysisĬell suspensions were isolated before two- or three-color immunofluorescence analysis. Single-cell suspensions isolated from the various compartments (spleen, lymph nodes, or BM) were counted using a Malassez cell chamber and brought to a concentration of 5 × 10 6 cells/ml in RPMI 1640 containing 2% FBS and 0.01% NaN 3. Lymph node lymphocytes were collected from cervical, axillary, inguinal, and portal mesenteric lymph nodes. Erythrocytes were lysed with Tris-buffered ammonium chloride. PeC cells were removed by aseptic injection of 5 ml of RPMI 1640 into the PeC, followed by withdrawal of the peritoneal exudate. Splenic cells were depleted of erythrocytes using Tris-buffered ammonium chloride. RBC were lysed with Tris-buffered ammonium chloride. For BM preparation, femurs and tibias were removed aseptically, ground in a mortar containing 1 ml of ice-cold sterile RPMI 1640 (BioWhittaker), and filtered through sterile cotton to remove bone fragments. Spleen, lymph node, bone morrow (BM) and peritoneal cavity (PeC) cells were also collected. Cells were pelleted and resuspended in PBS supplemented with 2% FBS and 0.01% NaN 3. Materials and Methodsįor preparation of lymphocytes from peripheral blood, mice were bled retro-orbitally in heparinized tubes, and heparinized blood (0.2 ml) was pelleted by centrifugation, after which the cell pellet was collected and viable white cells were isolated using MSL cell separation gradient (Eurobio). The data reveal a novel mechanism used by this B cell SAg to cripple humoral immunity. In the present study, we made use of transgenic mice expressing fully human Igs to investigate the in vivo consequences of the confrontation of SpA with B lymphocytes. ![]() In addition, there is little insight into the in vivo effects of SpA on B lymphocytes expressing human V H3 + Igs, partly because of the unavailability of an appropriate experimental system. aureus to subvert the humoral immune response. It remains unclear whether this property represents a virulence factor used by S. Because of its high specificity for human Igs encoded by V H3 + genes ( 6) (>50% of the Ab repertoire), SpA is potentially capable of impacting a significant proportion of the human B cell repertoire. With approximately half of the total pool of functional genes, the V H3 family is the largest family that imparts protective humoral responses against pathogens ( 4, 5). In humans, Ig V H genes have been categorized into seven families of sequence homologous members. ![]()
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